This laboratory is appropriately titled Translational Research, as we use inherited retinal degenerations identified in the clinic as both a source of clues about retinal function and dysfunction and a target for research in therapeutic intervention. The broad direction for our laboratory involves the biology of photoreceptor rescue and repair and opportunities to initiate human clinical rescue trials for RP and allied diseases based on animal studies. We have studied a number of mouse and rat models of human retinal degeneration diseases to elucidate the mechanisms of retinal neural signaling deficiencies and degeneration leading to blindness. We use normal rodents and rodents that are genetically altered to mimic human retinal disease to study the characteristics (phenotype), molecular genetics, physiological mechanisms and possible treatments of these inherited retinal degenerations. Our laboratory applies the techniques of light and electron microscopy, immunohistochemistry, protein and lipid biochemistry, and molecular biology to human and animal retinal tissue, as well as the electroretinogram (ERG), ocular coherence tomography (OCT) and behavioral measurements in living animals to access retinal structure and function in ways similar to those used to evaluate human vision in the clinic. These studies address human conditions of retinal and macular degenerations and age-related macular degeneration. Mechanisms of Retinal Degeneration: A critical facet of retinal neurodegenerative disease involves the structural changes, particularly to the photoreceptor outer segments (OS), that precede photoreceptor death, causing loss of vision. As photoreceptor cells undergo primary degeneration through progressive outer segment (OS) shortening in many of these conditions, a critical question is whether the outer segment may exhibit sufficient structural plasticity to support elongation of OS that have been shortened by disease states and whether this would promote survival of the photoreceptor cell. The goal of the work is to investigate the molecules that are important in the regulation of OS length under light stress and genetic degenerative conditions. We are focusing on neurotrophic factors, such as CNTF, and on small molecules that regulate cytoskeletal growth, including such as Rac1.In addition, a common pathway leading to cell death in many retinal degenerations is oxidative stress due to the generation of reactive oxygen species (ROS). Rac1 may play a role here too, as it is a critical component of the ROS generator NADPH oxidase. Role of Rac1 in retinal plasticity, development and oxidative damage: This year we continued a molecular approach to studying retinal disease mechanisms by investigating the role Rac1 in photoreceptor plasticity and cell death by oxidative stress in normal and diseased retinas using Rac1 transgenic and conditional knockout mice. Rac1 is a protein that can function as an intracellular molecular switch and is activated by various membrane receptors to produce a variety of downstream biological effects in many different cell types. We use a method called conditional gene targeting to modify the gene for Rac1 to learn about its role in photoreceptors. By this method only the gene in these cells is altered, leaving the Rac1 gene in other cell types unaffected. One of the photoreceptor specific functions of Rac1 in invertebrate photoreceptors is to regulate photoreceptor morphogenesis, and in particular the photoreceptive membrane analogous to outer segments in mammals. This was discovered using conditional gene targeting to produce depletion of Rac1 or constitutive activation of Rac1 in photoreceptors. Another of its functions in mammalian cells is activating NADPH oxidase, which can lead to oxidative damage. We showed that a 50% knockdown of Rac1 in mouse photoreceptors reduced oxidative damage due to excessive light exposure, but did not affect normal retinal structure or function. We published a study showing that NADPH oxidase activity accompanied by increased production of reactive oxygen species (ROS)contributes to photoreceptor degeneration in CA-Rac1 mice. This may be useful in understanding the mechanisms of some types of inherited or environmentally induced retinal degenerations and in designing treatments. Inhibiting the Rac1-NADPH oxidase pathway could be neuroprotective in a broad spectrum of retinal diseases To further explore the role of Rac1 in mammalian photoreceptor development and plasticity, we used conditional gene targeting to make a mouse which expresses a constitutively active form of Rac1 (CA-Rac1) in rod photoreceptors. Expression in photoreceptors coincided with that of the major outer segment protein rhodopsin, which allowed us to test its effect on postnatal development. CA-Rac1 had a profound negative effect on mouse rod cell viability and development. Rod photoreceptors in the CA RAC1 retina exhibited a defect in polarity and migration. CA-Rac1 disrupted rod morphogenesis and gave a phenotype resembling a form of retinitis pigmentosa. The outer segment portion of the displaced cells was either absent or severely shortened. Our study implicates an important role for RAC1 in the process of initial polarization and morphogenesis during rod photoreceptor development. Experiments are currently underway using CA-Rac1 in mouse models of photoreceptor degeneration to investigate a possible role of for Rac1 in maintaining photoreceptor outer segments is disease states. Retinoids: Retinoids are important for the light absorbing properties of photoreceptors. Abnormal processing of retinoids in the retina is responsible for several types of retinal degeneration. We are using mouse retinal disease models, the ABCA4 knockout mouse model of Stargardts macular degeneration and the RPE65 knockout mouse, which have abnormal retinoid processing, to test a strategy to retard degeneration. Retinoschisnin Structure and Function: Mutations in the gene for retinoschisin protein (RS1) found on the X chromosome cause X-linked retinoschisis (XLRS). XLRS is an inherited retinal disease and is a leading cause of juvenile macular degeneration in human males. The RS1 is found primarily on the outer plasma membrane of photoreceptor inner segments, where the energy and protein producing machinery of the cell reside . However, the role of RS1 in photoreceptor function is not known. We showed that young mice lacking retinoschisin have delayed maturation of outer segments. This may be related to changes we found in transcription factors, which determine the level of proteins involved in photoreceptor transduction. In another indication that RS1 is important in photoreceptor function, changes in mitochondrial structure observed using electron microscopy in mice lacking Rs1 before and after gene therapy were associated with changes seen in the OCT reflective bands in live animals. Thus, OCT may be useful in the clinic for tracking treatment of XLRS. RS1 protein is also present in the synaptic and inner nuclear layers of the retina where it plays a role in maintaining structure of the tissue and proper cell to cell signaling.We found that lack of RS1 affects the localization of proteins at the synaptic connection between photoreceptors and the next neuron in the chain passing visual information on to the brain. Dysfunction at this connection would help explain some of the vision loss and abnormal electrophysiological response in XLRS patients. Treating the Rs1-KO mouse model of XLRS with a vector delivering the missing gene partially restores the synaptic proteins to their normal location. We found that rearing mice lacking RS1 protein in dim light reduces inner retina pathology, suggesting that the neural activity induced by light causes structural and functional changes in the retina in the absence of RS1.